This invention relates to materials involved the control of the cardiovascular systems, and in particular, to activities mediated by thrombin and its cellular receptor.
2. References
The following references are cited in this application as superscript numbers at the relevant portion of the application:
1. Vu, T.-K. H., D. T. Hung, V. I. Wheaton, and S. R. Coughlin. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 64: 1057-1068 (1991). PA1 2. Coughlin, S. R., T.-K. Vu, D. T. Hung, and V. I. Wheaton. Characterization of a functional thrombin receptor. J. Clin. Invest. 89: 351-355 (1992). PA1 3. Vouret-Craviari, V., E. Van Obberghen-Schilling, U. B. Rasmussen, A. Pavirani, J.-P. Lecocq, and J. Pouyssegur. Synthetic .alpha.-thrombin receptor peptides activate G-protein-coupled signaling pathways but are unable to induce mitogenesis. Mol. Biol. Cell. 3:95-102 (1992). PA1 4. Zhong, C., D. J. Hayner, M. A. Corson, and M. S. Runge. Molecular cloning of the rat vascular smooth muscle thrombin receptor. J. Biol. Chem. 267: 16975-16979 (1992). PA1 5. Muramatsu, I., A. Laniyonu, G. J. Moore, and M. D. Hollenberg. Vascular actions of thrombin receptor peptide. Can. J. Physiol. Pharmacol. 70: 996-1003 (1992). PA1 6. deblois, D., G. Drapeau, E. Petitclerc, and Marceau, Francois. Synergism between the contractile effect of epidermal growth factor and that of des-Arg.sub.9 -bradykinin or of .alpha.-thrombin in rabbit aortic rings. Brit. J. Pharmacol. 105: 959-967 (1992). PA1 7. Vassallo, R. R., Jr., T. Kieber-Emmons, K. Cichowski, and L. F. Brass. Structure-function relationships in the activation of platelet thrombin receptors by receptor derived peptides. J. Biol. Chem. 267: 6081-6085 (1992). PA1 8. Chao, B. H., S. Kalkunte, J. M. Maraganore, and S. R. Stone. Essential groups in synthetic agonist peptides for activation of the platelet thrombin receptor. Biochemistry 31: 6175-6178 (1992). PA1 9. White, R. P., C. E. Chapleau, M. Dugdale, and J. T. Robertson. Cerebral arterial contractions induced by human and bovine thrombin. Stroke 11: 363-368 (1980). PA1 10. DeMey, J. G., M. Claeys, and P. M. Vanhoutte. Endothelium-dependent inhibitory effects of acetylcholine, adenosine triphosphate, thrombin and arachidonic acid in the canine femoral artery. J. Pharmacol. Exp. Ther. 222: 166-173 (1982). PA1 11. Rapoport, R. M., M. B. Draznin, and F. Murad. Mechanisms of adenosine triphosphate-, thrombin-, and trypsin-induced relaxation of rat thoracic aorta. Cir. Res. 55:468-479 (1984). PA1 12. White, R. P., Y. Shirasawa, and J. T. Robertson. Comparison of responses elicited by alpha-thrombin in isolated canine basilar, coronary, mesenteric, and renal arteries. Blood Vessels. 21: 12-22 (1984). PA1 13. Walz, D. A., G. F. Anderson, and J. W. Fenton. Responses of aortic smooth muscle to thrombin and thrombin analogues. Annals New York Acad. Sci. 485: 323-334 (1986). PA1 14. Hollenberg, M. D., S. -G. Yang, A. A. Laniyonu, G. J. Moore, and M. Saifeddine. Action of thrombin receptor polypeptide in gastric smooth muscle: Identification of a core pentapeptide retaining full thrombin-mimetic intrinsic activity. Mol. Pharmacol. 42: 186-191 (1992). PA1 15. Yang, S.-G., A. A. Laniyonu, M. Saifeddine, G. J. Moore, and M. D. Hollenberg. Actions of thrombin and thrombin receptor peptide analogues in gastric and aortic smooth muscle: Development of bioassays for structure-activity studies. Life Sciences, 51: 1325-1332 (1992). PA1 17. Vu, T.-K. H., V. I. Wheaton, D. T. Hung, I. Charo, and S. R. Coughlin. Domains specifying thrombin-receptor interaction. Nature 353: 674-677 (1991). PA1 18. Yang, S.-G., M. Saifeddine, A. Laniyonu, and M. D. Hollenberg. Distinct signal transduction pathways for angiotensin-II in guinea pig gastric smooth muscle: Differential blockage by indomethacin and tyrosine kinase inhibitors. J. Pharmacol. Exp. Ther. 264: 958-966, 1992. PA1 19. Muramatsu, I., H. Itoh, K. Lederis, and M. D. Hollenberg. Distinctive actions of epidermal growth factor-urogastrone in isolated smooth muscle preparations from guinea pig stomach: differential inhibition by indomethacin. J. Pharmacol. Exp. Ther. 245: 625-631 (1988). PA1 20. Savarese, T. M., and C. M. Fraser. In vitro mutagenesis and the search for structure function relationships among G protein-coupled receptors. Biochem. J. 283: 1-19 (1992). PA1 21. Dimaio, J., F. R. Ahmed, P. Schiller, and B. Belleau. Stereoelectronic control and decontrol of the opiate receptor. Recent Advances in Receptor Chemistry; F. Gualtieri, M. Giannella and C. Melchiorre, eds. 1979 Elsevier/North-Holland Biomedical Press. pp. 221-234 (1979). PA1 22. Ahlquist, R. P. A study of the adrenotropic receptors. Amer. J. Physiol. 153: 586-600 (1948). PA1 23. Barlos K., Gatos, D., Hondrelis, J., Matsoukas, J. M., Moore, G. J., Schafer, W. and Sotiriou, P. Preparation of new acid labile resins of the secondary alcohol type and their application in peptide synthesis. Liebigs Ann. Chem. 951-955 (1989). PA1 24. Matsoukas, J. M., Agelis, G., Hondrelis, J., Yamdagni, R., Wu, Q., Ganter, R., Smith, J., Moore, D. and Moore, G. J. Synthesis and biological activities of angiotensin II, sarilesin and sarmesin analogues containing Aze or Pip at position 7. J. Med. Chem. 36: 904-911 (1993). PA1 25. Matsoukas, J. M., Cordopatis, P., Belte, U., Goghari, M. N., Franklin, K. J. and Moore, G. J. Importance of the N-terminal domain of the type II angiotensin antagonist Sarmesin for receptor blockade. J. Med. Chem. 31: 1418-1421-(1988). PA1 26. Stewart, J. M. and Young, J. D. Solid Phase Peptide Synthesis, Second Edition. Pierce Chemical Company, Rockford, Ill. (1984).
16. Matsoukas, J. M., M. H. Goghari, M. N. Scanlon, K. J. Franklin, and G. J. Moore. Synthesis and biological activities of analogs of angiotensin II and III containing O-methyl-tyrosine and D-tryptophan. J. Med. Chem. 28: 780-783 (1985).
The disclosure of all publications, patents and patent applications are herein incorporated by reference in their entirety.
3. State of The Art Thrombin is a powerful factor in regulating the state of the cardiovascular system. It is clear that thrombin aids in the formation of blood clots by catalyzing the conversion of fibrinogen to fibrin, which is an integral part of most clots. In addition, thrombin is known to act directly on cells in the blood and on the interior blood vessel wall, and specifically to activate platelets to form clots. Thrombin-induced platelet activation is particularly important for arterial thrombus formation, a process that causes myocardial infarction and some forms of unstable angina and stroke. In addition, thrombin promotes inflammation and other cellular activities. Thrombin is chemotactic for monocytes, mitogenic for lymphocytes and smooth muscle cells, and causes endothelial cells to express the neutrophil adhesive protein GMP-140 on their surfaces and inhibits the growth of these cells. Thrombin elicits platelet-derived growth factor from the endothelium and is mitogenic for mesenchymal cells.
In addition to its role as a coagulation factor, thrombin is now known to regulate cell function via the proteolytic activation of a specific G-protein-linked cell surface receptor .sup.1-4. The novel mechanism whereby thrombin activates a target cell involves the proteolytic exposure of an N-terminal sequence of the receptor; this revealed N-terminal domain (beginning with Serine .sub.42 of the human receptor) is then believed to act as an "anchored" and "tethered" ligand that activates the receptor .sup.2. Remarkably, it has been demonstrated that a peptide comprising only the first 14 amino acids of the thrombin-revealed N-terminal sequence of the human receptor (abbreviated P14) can, on its own, activate the thrombin receptor, so as to mimic many of the cellular actions of thrombin in a variety of target tissues .sup.1,3,5-8. In our own work .sup.5, we have established that the human thrombin receptor-derived tetradecapeptide (P14, S.sub.42 FLLRNPNDKYEPF.sub.55, SEQ ID NO: 1) possesses thrombin-mimetic activity in both vascular and non-vascular smooth muscle preparations. In accord with the actions of thrombin itself .sup.9-13, we observed that P14 can cause either a direct contractile response to gastric or vascular smooth muscle .sup.5,4 or an endothelium-dependent relaxation of selected vascular smooth muscle preparations, such as the one derived from rat aortic tissue .sup.5.
We used the guinea pig and rat gastric longitudinal muscle (LM) strip contractile assays .sup.15 and the rat aortic (RA) ring relaxation assay as reliable and convenient bioassays to establish that only the first five amino acids of P14 (i.e.,P5, or S.sub.42 FLLR.sub.46, SEQ ID NO: 2) are required to mimic the actions of thrombin on smooth muscle preparations-.sup.14. In the pentapeptide (S.sub.42 FLLR.sub.46, SEQ ID NO: 2), we observed further that the C-terminal arginine-46 as well as phenylalanine-43 play critical roles in activating the thrombin receptor in the LM and RA tissues. The pentapeptide, P5, has also been shown to exhibit thrombin-like activity in fibroblasts .sup.3 and in platelets .sup.8. Structure-activity studies have demonstrated that in platelets as well, the phenylalanine-43 and arginine-46 residues are important for the biological activity of the receptor-derived polypeptides .sup.7,8.
There is a need for thrombin receptor activators and inhibitors having increased biological activity and/or resistance to degradation or metabolism.